New bioanalytical tools and devices: chemistry leads the way

Introduction

Three University of Alberta chemists are developing innovative and imaginative bioanalytical techniques aimed at tackling the burden and suffering caused by infectious diseases in the developing world.

Bioanalytical chemists are accustomed to having a plethora of convenient consumables and sophisticated instrumentation at their disposal. In the absence of this infrastructure it is hard to imagine how one might be able to achieve the primary goals of bioanalytical chemistry – the detection of small molecules or macromolecules in biological systems. Unfortunately, many researchers in resource-limited developing countries, or field workers in remote locations far from modern conveniences, are unable to take advantage of modern bioanalytical techniques due to a lack of infrastructure. Compounding the misfortune of this situation is that these same researchers are those who have the greatest need for rapid and accurate bioanalytical detection methods, typically to diagnose diseases such as tuberculosis or malaria, which are a disproportionate burden on the developing world.

“By keeping the assay simple and minimizing the complexity of the technology, we are hopeful that this will be more easily adopted in resource-limited settings,” says Dr. Julianne Gibbs-Davis.

New bioanalytical techniques designed from the bottom up to address the needs of researchers in resource-limited environments could help level the playing field and facilitate point-of-care diagnosis in the developing world. Motivated by this shared goal, Dr. Julianne Gibbs-Davis, Dr. Ratmir Derda, and Dr. Michael Serpe in the Department of Chemistry are, independently, taking three very different approaches towards the development of new bioanalytical tools and devices. Each of these researchers is effectively leveraging the cutting edge infrastructure of a world class research institution to lower the financial threshold limiting access to modern bioanalytical chemistry.

Move over PCR!

The polymerase chain reaction (PCR) is the method of choice for detecting low concentrations of DNA and is routinely used for the detection and diagnosis of viral and bacterial diseases. However, PCR requires the use of thermal cycler instrumentation that may not always be available in resource limited laboratories or sites that lack reliable access to electricity. In such situations, it would be preferable to use an instrument-free method to amplify the concentration of DNA in a dilute sample until reaching a concentration where it can be easily detected using common fluorescence-based methods. One potential application would be the inexpensive diagnosis of tuberculosis by detection of characteristic DNA sequences from the causative agent, Mycobacterium tuberculosis.

In an effort to reach this goal, Gibbs-Davis turned to an idea that she had been eager to try since her days as a graduate student at Northwestern University. Briefly, Gibbs-Davis envisioned a system in which one strand of DNA would template the enzymatic ligation of two partially complementary fragments. Normally, such a reaction would produce a double stranded DNA duplex that would be substantially more stable than the free single strand DNA pieces and thus represent the end of the reaction process. Gibbs-Davis’s ‘eureka’ moment insight came when she realized that she could engineer the system such that the DNA duplex was destabilized enough that it would occasionally dissociate to form the single stranded pieces (Figure 1). This dissociation frees the template strand to engage in another cycle of ligation, essentially acting as a catalyst for the ligation reaction of the two complementary fragments. In their initial versions of this design, Gibbs-Davis and her coworkers succeeded in coaxing a template strand into performing 18 cycles of ligation, or turnovers, in 20 hours, a substantial improvement over previously reported ligation-based amplifications under similar conditions.¹

The first generation of Gibbs-Davis’s amplification system detects small DNA fragments that are complementary to the template sequence. To detect a longer and intact target DNA sequence, Gibbs-Davis devised a cross-catalytic ligation cycle in which a target DNA sequence, necessarily composed of unmodified DNA, ligates two partially complementary strands to make a new destabilizing template that can participate in the catalytic ligation of additional fragments. In this way a piece of target DNA acts as a trigger for initiating the catalytic cycle of DNA amplification. With the latest version of her cross-catalytic ligation system, Gibbs-Davis can now get thousands of copies of template molecule in just a few hours of incubation. While this is still not approaching the billions of copies that can be obtained from PCR amplification, it is already practical for some applications, and further improvements are likely.

Gibbs-Davis plans to incorporate this amplification system into an inexpensive kit, which will include a sample holder with a built in light emitting diode for fluorescence detection, for diagnosis of tuberculosis and multi-drug resistant tuberculosis.  As Gibbs-Davis explains, “By keeping the assay simple and minimizing the complexity of the technology, we are hopeful that this will be more easily adopted in resource-limited settings.”  With continued optimization, she expects that it will be possible to get the reagent costs under a few dollars per test, making her method cost competitive with traditional PCR-based methods.

Origami petri dish 

Compared to the traditional animal shapes and intricate geometric forms produced by the Japanese art of origami, a petri dish seems to be a particularly unlikely structure to create from a sheet of paper. However, this has not stopped Dr. Ratmir Derda from developing a petri dish replacement from a sheet of paper that has been cleverly folded and manipulated to create a sterile environment for cell growth. Such devices could enable researchers in the developing world to carry out cell-based bioanalytical assays for research purposes or for detection of disease-causing agents.

To achieve the goal of creating a paper-based cell culture system, Derda has abandoned the paradigm of growing bacteria in an open dish (i.e., a petri dish) with a loose fitting lid. Rather, he has designed a self-contained system in which bacteria grow inside a sterile paper envelope that retains liquid growth media, yet is permeable to oxygen (Figure 2). This growth chamber has a transparent window such that a researcher can easily visualize the growth of the bacteria inside. A particularly effective strategy for facilitating the visualization of bacteria growth in the chamber is to introduce a gene for a brilliantly coloured red fluorescent protein. In this way, the amount of bacterial growth can be qualitatively assessed by eye, or quantitatively assessed using a camera phone and a custom software application.

In tackling the problem of how to most effectively facilitate cell culture techniques in resource-limited environments, Derda endeavoured to find a convenient testing ground. His inspiration was to turn to Canadian high school classrooms, which, relative to chemistry labs at the University of Alberta, represent a resource-limited environment that could approximate the level of infrastructure one could expect to find in a lab in the developing world. Accordingly, Derda has been working closely with high school students to optimize the procedure for mass production of his paper-based petri dishes.

With the support of a Canada’s Rising Stars in Global Health grant from Grand Challenges Canada, Derda is exploring the combined use of paper-based petri dishes and engineered bacteriophage for biosensing applications. One of his primary goals is to use this system for detecting tuberculosis-specific antibodies in serum and he states that “the first functional prototype of the phage-based diagnostic could be ready to showcase after a few months”. Over the longer term, he foresees application to a broader array of bioanalytical applications, such as rapid and inexpensive diagnostics for HIV or malaria.

Derda has also used a substantial portion of his Rising Star award to organize a workshop in Kenya that will bring together like-minded researchers who are working towards the development of diagnostic devices for use in resource-limited environments. Derda hopes that, by holding the conference in Africa, he will have the opportunity to give a number of North American researchers first hand experience of the actual level of resources available in these ‘low resource’ environments. When asked about his own recent trip to Kenya, Derda replied, “Many laboratories I saw were ghost labs filled with equipment that cannot be used because the lab does not have consumables”. Apparently, programs that redistribute surplus equipment from well-funded labs have been very successful, but the problem is that much of this equipment relies on manufactured consumables that simply are not available. The solution, Derda says, is the concept of “on-site production”. Specifically, if researchers in developing countries could produce low-cost alternatives to common laboratory consumables they would be in a much better position to take advantage of the instrumentation resources that are available to them.

Technicolor bioanalysis

While not every lab in the developing world can afford a visible wavelength spectrophotometer, most humans have a built-in equivalent: their colour vision. The key to taking advantage of human colour perception for bioanalysis is having a robust method for converting the concentration of a target analyte into a visible colour change. Inspired by the brilliant blue wings of a butterfly, the Serpe group is now developing devices that achieve this goal.

The surface of the Morpho didius butterfly wing is covered in small ridges that have just the right spacing to cause constructive interference of blue light, while other wavelengths are removed by destructive interference. In principle, if the butterfly was able to change the spacing of the ridges on its wings it could change its colour to green or red by causing constructive interference of the appropriate wavelengths of visible light. Although butterfly wings do not have this capability, the Serpe group is now building devices that are artificial mimics of the butterfly wing, yet are capable of changing their colour in response to a biomolecular target analyte.²

To create these colour-changing biosensors, Serpe has turned to etalons: a sandwich structure composed of two reflective gold layers (the bread) on either side of a layer of microgel particles (the filling) (Figure 3). At a certain thickness of filling, the device appears a certain color due to the constructive interference of particular wavelengths of light. However, when the particles are induced to change their size, the thickness of the filling changes and the colour of the device undergoes a corresponding change. Serpe explains that “…the polymer can be made such that it interacts with, and binds to, biomolecules that are indicative of disease or infection. Hence, the device’s colour change can be coupled to the presence of a specific biomarker for disease, and used as a diagnostic device.” Serpe has already demonstrated that this principle can be applied to glucose sensing³ and, with the support of a Canada’s Rising Stars in Global Health grant from Grand Challenges Canada, is now working to extend the approach to a wider range of bioanalytical target molecules. Short-term goals include the detection of biomarkers for malaria and tuberculosis, while longer-term goals involve extending this technology to the detection of biomarkers for the early stages of Alzheimer’s disease.

Canadian chemistry leads the way

This article has highlighted three exciting new approaches towards enabling robust bioanalytical detection methods that do not require the cutting edge infrastructure and modern conveniences that have helped Canada become a world leader in bioanalytical research. With the seemingly unlimited resource of researcher ingenuity, and the continued generous support of funding agencies such as Grand Challenges Canada, Canadian researchers are well positioned to play a key role in the uplifting of nations who do not share our advantages.

References

1. Kausar, A.; McKay, R.D.; Lam, J.; Bhogal, R.S.; Tang, A.Y.; Gibbs-Davis, J.M., Angew. Chem. Int. Ed. Engl. 2011, 50(38), 8922-8926.
2. Hu, L.; Serpe, M.J., Polymers 2012, 4(1), 134-149.
3. Sorrell, C.D.; Serpe, M.J., Anal. Bioanal. Chem. 2012, 402(7), 2385-2393.